HPV Epidemiology
The correlation between the potential for development of cervical carcinoma and the presence of Human Papilloma Virus (HPV) infection has become well established. For example, in a worldwide survey of cervical carcinomas, 93% of the specimens showed evidence of the presence of HPV sequences (Bosch et al., 1995 J Nat Cancer Inst 87; 796-802). Since PCR primers used for that study were derived from the L1 region, some of these specimens were retested with primers from other regions and a rate of 99.7% was reached (Walboomers et al. 1999 J Path 189; 12-19) demonstrating that in all likelihood the presence of HPV is a necessary condition for development of cervical carcinoma.
However, although physically and phylogenetically related, HPV does not represent a homogeneous population of viruses; there are a large number of different HPV types that differ from each other with regard to nucleic acid homology and properties such as tissue tropism and oncogenic potential. For instance, certain HPV types can be grouped together on the ability to infect genital-mucosa tissues and another group of HPV types can be formed that is linked together by the ability to carry out cutaneous infections. For the genital-mucosal trophic HPV, a survey of which particular HPV types are present in specimens with various levels of tumor progression allows risk assessment for cervical carcinoma to be carried out for any given HPV type. Thus, a demarcation has been drawn between genital-mucosa HPV types that are unlikely to lead to an oncogenic state (the Low Risk group) and HPV types that exhibit a significant risk of tumor progression (the Medium and High Risk groups). This is more than of academic interest since the presence of the Medium or High Risk group can have prognostic value that can direct the treatment of the patient. Various papers have been written concerning the value of HPV testing with regard to the nature of the patient, the nature of a specimen and the particular type of HPV that is identified as being present.
For instance, the HPV Hybrid Capture II test, a commercially available FDA approved assay (Digene, Inc. Rockville, Md.) provides two cocktails of RNA probes: a Low Risk group for detection of HPV 6, 11, 42, 43 and 44 and a High Risk group for detection of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Signal generation is carried out by labeled antibodies that have specificity for RNA/DNA hybrids. The presence or absence of the low risk group did not seem to generate information that could be used to alter the treatment of patients whereas positive results from the High Risk group did have diagnostic and prognostic value. As such, this test was later modified and approved by the FDA such that only the cocktail for the High Risk group could be used. This method suffers from the need to use a large number of separate probes simultaneously (13 in the High Risk cocktail alone). Early on it was recognized that the majority of HPV positive cancers seemed to have either HPV 16 or HPV 18, while each of the other HPV types seemed to be present in a much smaller number of cases. Individually, each of these other types is a “minor” causative agent, but as a group they can collectively represent a major risk factor in the likelihood of development of cervical carcinoma. As such, the addition of other HPV types besides 16 and 18 can add incremental levels of sensitivity of detection of oncogenic HPV.
However, the use of a probe cocktail that contains so many individual species has the result that the concentration of each individual probe has to be maintained at a sufficient level that it can drive the reaction for hybridization with its appropriate target. The presence of such an increase in complexity of sequence information in the probe mix and the presence of such a heightened amount of probe in the hybridization mixture can lead to cross-reaction with other HFV types and non-specific binding. This may explain why the High Risk cocktail exhibited signals with HPV 6, 11, 40, 42, 53, 55, 70 and MM8, HPV types that are not considered to be associated with cancer development (ALTS Group 2000 J. National Cancer Inst. 92; 397-402; Hughes et al. 2002 Am J Obstet Gynecol 186; 396-403). Thus, the increased breadth that was implemented in this assay may also have conveyed a negative quality of a broader spectrum of sensitivity to non-oncogenic HPV as well.
Amplified Assays
Sensitivity and selectivity can also be enhanced by the use of nucleic acid amplification technology. Exponential amplification as first exemplified in PCR by Mullis et al., (U.S. Pat. No. 4,683,195 hereby incorporated by reference) is achieved by the use of at least one pair of primers: a forward primer and a reverse primer. These primers can be defined in various ways. For instance, in terms of a double stranded target molecule, a forward primer comprises sequences complementary to one strand and a reverse primer comprises sequences complementary to the other strand. In terms of a single strand, a forward primer comprises sequences complementary to one region of a target nucleic acid strand and the reverse primer comprises sequences identical to a different region of the target nucleic acid strand. Due to this arrangement, a forward primer binds to a complementary target molecule followed by extension using the target as a template, thereby providing a copy of a portion of the target molecule (the forward reaction). The reverse primer uses this copy as a template for another binding and extension reaction (the reverse reaction) thereby providing another copy that can be used by a forward primer and so on. Thus, a series of forward and reverse copying reactions provides amplification of the portion of nucleic acid sequences between the binding sites for the forward and reverse primers.
Type Specific Amplification
Exploiting this methodology, selective amplification of various HPV types has been carried out by the design of PCR primer pairs that are specific for each type of HPV. Conditions can be established such that amplification takes place only when a particular HPV target is present in a specimen. The resultant product can then be evaluated by the presence of an amplification event itself as judged by gel analysis or by incorporation. Further specificity can be insured by using type specific probes and detecting the presence of individual HPV sequences in the amplified products.
This method has practicality if only a limited number of types are being evaluated; for instance, if only the presence of HPV 16 and HPV 18 are being ascertained. However, as described above, inclusion of more HPV types may be needed to provide more sensitivity of HPV detection. Thus to generate the equivalency of the assay above, 13 separate primer pairs would be needed. Therefore, this method has the limitation that if individual amplifications are carried out, a large number of different type specific reactions are required to cover each oncogenic type that is desired to be included in an assay. This decreases the amount of sample available for each reaction and increases the amount of reagents and supplies for the cost of the test. As such, multiplex amplification is usually done with a more limited number of different potential targets since the addition of numerous individual primers of each HPV type increases the total amount of primers in the reaction mixture thereby encouraging non-specific priming events. An example of this is a multiplex amplification of 6, 11, 16 and 18 (Anceschi et al., 1990 J. Virol Methods 28; 59-66). As described above for probe specific assays, this method is also self-defining in that the primers are designed to be capable of only amplifying a particular HPV type and other HPV types that may be present are not amplification targets.
Consensus Primers for Amplified Assays
Systems for the generic amplification of multiple species of HPV have also been carried out by a number of groups. As described above, it has been used to establish the correlation between HPV infection and the development of cervical cancer. Although the definition of different types of HPV is based on differences in sequences in homology, the sequence variation is not homogeneous over the length of the viral genome and two particular genes, E1 and L1, tend to be more conserved than other segments. Although no particular sequence is completely preserved within the genomes of all the various HPV types, relatively conserved sequences can be found. A consensus sequence can be used for forward and reverse primers and amplification carried out under conditions where a certain level of mismatches is tolerated (van den Brule et al., 1990 Int J. Cancer 45; 644-649). Another example was recently presented by Kleter et al., 1999 (J Clin Micro 37; 2508-2517) which used sequences derived from HPV 16 as primers. This system was able to amplify HPV samples from types that had as many as 4 mismatches by using relatively non-stringent conditions of 52° C. as an annealing temperature. Alternatively, to maximize the spectrum of HPV types that can be used as substrates, the primers can be designed such that at variable positions, an indiscriminate base such as Inosine (Gregoire et al., 1989 J. Clin Micro. 27; 2660-2665) or a mixture of bases can be used (Manos et al., 1989 Cancer Cells 7; 209-214). Even with this design, there may still be a certain number of mismatches due to the diversity of sequences in different HPV types but conditions can be adopted such that amplification still takes place. Consideration of which particular types are the targets can also affect the primer design and amplification conditions. For instance, the GP 5,6 consensus primers used by van der Brule et al, 1990 (op. cit.) were designed to have no more than two mismatches with genital mucosa types 6, 11, 16, 18 and 31 but were allowed to have numerous mismatches with cutaneous varieties of HPV (van den Brule et al., 1990 Int J. Cancer 45; 644-649). A similar strategy was employed by Evander and Wadell (1991 J Vir. Methods 31; 239-250) where genital mucosa types 6, 11, 16, 18, 31 and 33 had a maximum of three mismatches and amplified efficiently with a 60° C. annealing temperature. As the annealing temperature was lowered to 55° C., HPV types 13 and 30 which are associated with oral and genital/oral tropisms respectively were efficiently amplified and faint bands were seen for cutaneous HPV types 2, 3 and 7. Thus the selectivity of the amplification reaction could be adjusted for the particular breadth that was desired.
As expected, the design of such general primers has allowed the amplification of the HPV types whose sequences were used to design these primers. However, the generality of these primers was also shown by the ability to amplify related HPV types which had been previously isolated and characterized but had not been sequenced at the time the primers were developed. For instance, in the paper by Evander and Wadell (supra), their primers were able to efficiently amplify HPV 13 and HPV 30 even though the sequences for these types only became available years afterwards. Amplification with consensus HPV primers has the advantage that its relative insensitivity to the nature of the particular HPV type in a specimen allows epidemiological surveys to be carried out without delineating which particular HPVs are defined as targets. However, due to the general nature of these amplification systems, the presence of an amplified product only generates the information that there was HPV in the specimen being tested. For epidemiological surveys or other purposes, the knowledge of the particular type of HPV has to be ascertained by additional analytical methods. For example, the amplification products can be used for RFLP analysis to identify characteristic patterns of restriction enzyme digestion (Lungo et al., 1992 Mol. Cell. Probes 6; 145-152). The amplified material can also be hybridized with type specific probes in a manner similar to that used for unamplified HPV genomic nucleic acids. For example, Jacobs et al., (2000 Int J. Cancer 87; 221-227) provides a list of 37 different probe sequences that can be used to identify any one of 37 different HPV types that could be amplified by the GP5+ GP6+ pair of consensus primers. Or if there is still material remaining, consensus positive specimens can be used in a secondary round of amplification with type specific primers.
The general nature of these amplification systems may be useful for determining which particular HPV types are associated with the malignancy state. This may then be used for the design of assays for selected HPV types such as the system described previously for Digene. For example, the use of consensus sequence primer amplification revealed that although HPV 45 is relatively rare in the United States (Lorincz et al., 1992 Obstet and Gynecol 79; 328-337), it had a high prevalence rate in samples from patients with severe dysplasia in Jamaica (Rattrey et al., 1996 J. Inf Dis 173; 713-721). Furthermore, although these generic systems are derived from comparisons between different sequences of known HPV types, the open nature of its amplification doesn't relegate it to only these types and consequently, novel HPV types are also candidates for amplification and detection. These novel types can then be isolated and characterized further. In contrast, a probe specific type of assay such as the one by Digene can detect multiple HPV types but it is not open-ended since the results are shaped by the decision of which particular HPV types are included in the probe cocktail.
As described previously, detecting the presence of HPV in general is not adequate since the nature of the particular HPV type may have a bearing upon the course of treatment. In one approach to this, Silverstein et al., (U.S. Pat. No. 5,888,724) have disclosed a method for the specific amplification and detection of oncogenic HPV. Primers were designed such that they had high homology with oncogenic types of HPV and low homology with types that were considered to have a low risk of oncogenic potential (HPV 6, 11, 30, 32, 34, 42 and 53). However, this assay has the limitation that their disclosure specifically points out that the sequences of two other HPV types, HPV 51 and HPV 52, were sufficiently different that they would not be amplified or detected in their system. Thus, although both of these types are usually considered to be bona fide members of the high risk group, the assay disclosed by Silverstein et al., has been designed to ignore the presence of two types of HPV that are considered to have high oncogenic potential. Also, the prevalence of different HPV types in cancers may have geographical differences. For instance, HPV 52 together with HPV 58 was seen to actually have a higher representation in cervical carcinomas than HPV 16 and HPV 18 in a survey of Chinese women (Huang et al., Int J Cancer 70; 408-411), thus a clinically important variety of HPV (HPV 52) was ignored by the Silverstein assay.
Additionally, the primers used for this method were only 16-mers. As such, the conditions used for amplification included an annealing step of 42° C. (near the Tm of the primers) followed by an extension step of 72° C. (where the thermostable polymerase can function efficiently). Thus, during the transition from the annealing to the extension temperatures, this process depends upon primers being extended before they can separate from their templates. However, due to the inefficiency of the polymerase at lower temperatures, it is likely that there are many instances of primers annealed to their appropriate target who are denatured before an extension event can go forward thereby reducing the efficiency of amplification. Thus, the method of Silverstein has the limitation that it lacks sufficient breadth to generate signals from a clinically significant HPV type and relies on short thermolabile primers to carry out their process.
The breadth of inclusivity or non-inclusivity of an assay can influence its utility or potential use. Thus, as described above, the Silverstein method of assaying for oncogenic types of HPV suffers from an inability to recognize certain oncogenic types of HPV that are clinically important. As described previously, in addition to HPV 16 and 18, HPV 45, 52 and 58 are oncogenic HPVs that would be important in being recognized by a clinical assay. On the other hand, there can be signal generation from HPV types that were not intended as targets. For instance, as described previously, the Digene assay inadvertently generated signals from some HPV types that were not included in the High Risk probe mixture (ALTS group 2000, op. cit. and Konya et al., J Clin Micro 38; 408-411). This is especially problematic with a cross reaction with HPV 6 since it is a highly prevalent HPV type that may be present in high numbers in a clinical specimen but is unlikely to lead to an oncogenic event. In some cases, due to their rarity and the presence of an oncogenic potential, the presence of signal generation would not materially affect the worth of the assay. An example of this would be HPV 30 which is among the unintentional targets of the Digene assay. Due to its rarity, it's not usually included among the group of oncogenic types but at the same time, it should be noted that it was originally isolated from a carcinoma (Kahn et al., 1986 Int J Cancer 37; 61-65).
The problem with these rare types is that the limited available data makes it difficult to properly assign risk assessments for these types. For example, the literature contains numerous articles where HPV 66 is included as a member of the High Risk group and other papers have it listed with the Low Risk group. HPV 66 should probably be included with the High Risk groups since the original isolation was from an invasive carcinoma (Tawhed et al., 1991 J. Clin Micro 29; 2656-2660). An attempt to study the oncogenic potential of some of these other types was carried out by Meyer et al., (1998 J Inf Dis 178; 252-255) where, among the members shown above, they concluded that MM4 and HPV 66 should probably be considered High Risk types and HPV 53 should be included among the Low Risk group despite its phylogenetic kinship with other oncogenic types. A followup study by this group (Meyer et al., 2001 Int J Gynecol Cancer 11; 198) also concluded that HPV 53 was unlikely to be oncogenic. This may be problematic in the Digene test since HPV 53 has been shown to be one of the types that is inadvertently picked up by the Digene assay (ALTS group 2000, op. cit. and Konya et al., op.cit.).